Pyridoxal 5'-phosphate (PLP) is an essential, ubiquitous coenzyme that participates in many aspects of amino acid and cellular metabolism. The long term-goal of this continuing project is to determine how PLP biosynthesis is regulated at the pathway and genetic levels and integrated into general cellular metabolism. E coli is an ideal model system for these physiological, genetic, and biochemical studies, because it synthesizes a form of the PLP-precursor, pyridoxine (PN; vitamin B6), like plants and certain other microorganisms, and can convert PN, pyridoxal (PL), and pyridoxamine (PM) into PLP by a scavenger pathway present in all organisms. Three Specific Aims will be pursued in this five-year proposal. (I)We will continue to investigate the most important unresolved issues concerning the pathway and biochemistry of PLP biosynthesis. The following hypotheses will be tested. (i) The epd(gapB) gene is required for PLP biosynthesis. (ii) The missing branch of PLP biosynthesis involves redundant transketolase activities that lead to D-deoxyxylulose formation. (iii) Purified serC(pdxF) transaminase uses two different substrates leading to PLP or L-serine biosynthesis. (iv) The pdxA and pdxJ gene products close the pyridine ring of PN. A corollary of this hypothesis is that PL/PM/PN kinase functions in the scavenger and not the de novo PLP biosynthetic pathway. (v) Conversion of PLP to PL occurs by a specific intracellular phosphatase, analogous to the one recently found in mammals. (II) We will continue to develop genetic approaches to study the regulation and function of the PLP biosynthetic pathway. Two hypotheses will be tested. (i) An alternative branch detected in pdxB, serC(pdxF), or tktA tktB mutants does not contribute significantly to de novo PLP biosynthesis in wild-type bacteria. (ii) Mutants lacking pathway and genetic regulation can be isolated in which intracellular PN and PLP concentrations are increased, thereby restoring the functions of mutant (PLP-KM) enzymes that bind PLP poorly. (III) We will continue to investigate the structure, expression, regulation, and coordination of the pdx biosynthetic genes, all of which are members of complex superoperons. This Aim has two parts. (i) We will examine the expression patterns of the pdx genes as a group to learn whether they are coordinately regulated by common mechanisms, including LRP, CRP, and growth-rate control, or in response to certain physiological conditions. (ii) We will study certain interesting problems in the regulation of specific pdx genes. The continuation of this project is important for several reasons. It is providing basic knowledge and insights into topics of fundamental biological interest, including the regulation and enzymology of coenzyme biosynthesis, the structure and regulation of complex superoperons, the evolution of biosynthetic pathways, and the function of important classes of enzymes, such as flavoprotein oxidases. This project has led to significant new information about mechanisms that integrate coenzyme biosynthesis into general cellular metabolism. Finally, this project is of biomedical and biotechnological relevance, because PLP plays well- documented, diverse roles in human intermediary metabolism, physiology, and disease.